Justin P. Annes M.D., Ph.D.

Assistant Professor of Medicine (Endocrinology)

Medicine - Endocrinology, Gerontology, & Metabolism

Bio

Bio

Dr. Justin Annes specializes in the treatment of hereditary endocrine disorders with particular focus on neuroendocrine-related conditions.He developed the Stanford Endocrine Genetics Clinic in 2012 which is part of the interdisciplinary Stanford Hypertension Center and Stanford Neuroendocrine Tumor Program. He has medical practice has focused on hereditary endocrine disease since 2008.

Links

Research & Scholarship

Current Research and Scholarly Interests

DIABETESThe prevalence of diabetes is increasing at a staggering rate. By the year 2050 an astounding 25% of Americans will be diabetic. The goal of my research is to uncover therapeutic strategies to stymie the ensuing diabetes epidemic. To achieve this goal we have developed a variety of innovate experimental approaches to uncover novel approaches to curing diabetes.

(1) Beta-Cell Regeneration: Diabetes results from either an absolute or relative deficiency in insulin production. Our therapeutic strategy is to stimulate the regeneration of insulin-producing beta-cells to enhance an individual?s insulin secretion capacity. We have developed a unique high-throughput chemical screening platform which we use to identify small molecules that promote beta-cell growth. This work has led to the identification of key molecular pathways (therapeutic targets) and candidate drugs that promote the growth and regeneration of islet beta-cells. Our goal is to utilize these discoveries to treat and prevent diabetes.

(2) The Metabolic Syndrome: A major cause of the diabetes epidemic is the rise in obesity which leads to a cluster of diabetes- and cardiovascular disease-related metabolic abnormalities that shorten life expectancy. These physiologic aberrations are collectively termed the Metabolic Syndrome (MS). My laboratory has developed an original in vivo screening platform t to identify novel hormones that influence the behaviors (excess caloric consumption, deficient exercise and disrupted sleep-wake cycles) and the metabolic abnormalities caused by obesity. We aim to manipulate these hormone levels to prevent the development and detrimental consequences of the MS.

HEREDIATY PARAGAGLIOMA SYNDROMEThe Hereditary Paraganglioma Syndrome (hPGL) is a rare genetic cancer syndrome that is most commonly caused by a defect in mitochondrial metabolism. Our goal is to understand how altered cellular metabolism leads to the development of cancer. Although hPGL is uncommon, it serves as an excellent model for the abnormal metabolic behavior displayed by nearly all cancers. Our goal is to develop novel therapeutic strategies that target the abnormal behavior of cancer cells. In the laboratory we have developed hPGL mouse models and use high throughput chemical screening to identify the therapeutic susceptibilities that result from the abnormal metabolic behavior of cancer cells.

As a physician scientist trained in clinical genetics I have developed expertise in hereditary endocrine disorders and devoted my efforts to treating families affected by the hPGL syndrome. By leveraging our laboratory expertise in the hPGL syndrome, our care for individuals who have inherited the hPGL syndrome is at the forefront of medicine. Our goal is to translate our laboratory discoveries to the treatment of affected families.

Abstract

Conducting polymers present an opportunity for developing programmable, adjustable, spatially, and temporally controllable drug delivery systems. While several small molecule drugs have been released from thin conductive polymeric films successfully, delivering large molecule therapeutics, such as polypeptides and nucleic acids, has remained a significant challenge. Poor drug loading (?ng cm(-2)) of thin films coupled with film instability has, in many cases, made conducting polymer films refractory to clinical development. To address these limitations, we have utilized conductive polymer nanoparticulate backbones to controllably release insulin, a high molecular weight, clinically relevant polypeptide. We find that the interaction between insulin and the polymer scaffold can be described by a simple Langmuir-type adsorption model. By modifying the ratio of the amount of nanoparticles to the amount of insulin, we have obtained drug loading percentages estimated to be as high as 51 wt% percent. In vivo experiments in mice confirmed retained bioactivity of the released insulin after electrical stimulation.

Abstract

We have identified a novel role for hyaluronan (HA), an extracellular matrix (ECM) polymer, in governing the mechanical properties of inflamed tissues. We recently reported that insulitis in type 1 diabetes (T1D) of mice and humans is preceded by intra-islet accumulation of HA, a highly hygroscopic polymer. Using the DORmO double transgenic (DO11.10 x RIPmOVA) mouse model of T1D, we asked whether autoimmune insulitis was associated with changes in the stiffness of islets. To measure islet stiffness, we used atomic force microscopy (AFM) and developed a novel "bed of nails"-like approach that uses quartz glass nanopillars to anchor islets, solving a long-standing problem of keeping tissue-scale objects immobilized while performing AFM. We measured stiffness via AFM nanoindentation with a spherical indenter and found that insulitis made islets mechanically soft compared to controls. Conversely, treatment with 4-methylumbelliferone (4-MU), a small-molecule inhibitor of HA synthesis, reduced HA accumulation, diminished swelling, and restored basal tissue stiffness. These results indicate that HA content governs the mechanical properties of islets. In hydrogels with variable HA content we confirmed that increased HA leads to mechanically softer hydrogels, consistent with our model. In light of recent reports that the insulin production of islets is mechanosensitive, these findings open up an exciting new avenue of research into the fundamental mechanisms by which inflammation impacts local cellular responses.

Abstract

Loss of insulin-producing ?-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet ?-cells remains an attractive strategy. ?-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain ?-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify ?-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a ?-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on ?-cells and the molecular mechanisms that regulate ?-cell growth.

Abstract

Loss of ?-cell mass is a cardinal feature of diabetes. Consequently, developing medications to promote ?-cell regeneration is a priority. 3'-5'-Cyclic adenosine monophosphate (cAMP) is an intracellular second messenger that modulates ?-cell replication. We investigated whether medications that increase cAMP stability or synthesis selectively stimulate ?-cell growth. To identify cAMP stabilizing medications that promote ?-cell replication we performed high-content screening of a phosphodiesterase-inhibitor (PDE-I) library. PDE3,4 and 10 inhibitors, including dipyridamole, were found to promote ?-cell replication in an adenosine receptor-dependent manner. Dipyridamole's action is specific for ?-cells and not ?-cells. Next we demonstrated that norepinephrine (NE), a physiologic suppressor of cAMP synthesis in ?-cells, impairs ?-cell replication via activation of ?2-adrenergic receptors. Accordingly, mirtazapine, an ?2-adrenergic receptor antagonist and antidepressant, prevents NE-dependent suppression of ?-cell replication. Interestingly, NE's growth-suppressive effect is modulated by endogenously expressed catecholamine-inactivating enzymes (COMT and MAO) and is dominant over the growth-promoting effects of PDE-Is. Treatment with dipyridamole and/or mirtazapine promote ?-cell replication in mice and treatment with dipyridamole is associated with reduced glucose levels in humans. This work provides new mechanistic insights into cAMP-dependent growth regulation of ?-cells and highlights the potential of commonly prescribed medications to influence ?-cell growth.

Abstract

The diabetes pandemic incurs extraordinary public health and financial costs that are projected to expand for the foreseeable future. Consequently, the development of definitive therapies for diabetes is a priority. Currently, a wide spectrum of therapeutic strategies-from implantable insulin delivery devices to transplantation-based cell replacement therapy, to ?-cell regeneration-focus on replacing the lost insulin-producing capacity of individuals with diabetes. Among these, ?-cell regeneration remains promising but heretofore unproved. Indeed, recent experimental work has uncovered surprising biology that underscores the potential therapeutic benefit of ?-cell regeneration. These studies have elucidated a variety of sources for the endogenous production of new ? cells from existing cells. First, ? cells, long thought to be postmitotic, have demonstrated the potential for regenerative capacity. Second, the presence of pancreatic facultative endocrine progenitor cells has been established. Third, the malleability of cellular identity has availed the possibility of generating ? cells from other differentiated cell types. Here, we review the exciting developments surrounding endogenous sources of ?-cell production and consider the potential of realizing a regenerative therapy for diabetes from adult tissues.

Abstract

The renin-angiotensin-aldosterone system (RAAS), vitamin D, and parathyroid hormone have all been implicated as regulators of adipocytokines and inflammation. We evaluated human interventional study protocols to investigate whether controlled modulations of these calcium- and sodium-regulatory hormones could influence adipocytokines and inflammation in obesity and diabetes.Post-hoc analyses of two separate human protocols (Protocol 1, n=14; Protocol 2, n=24) conducted in a clinical research setting after rigorous control of diet, posture, medications, and diurnal rhythm, were performed. Protocol 1 evaluated obese hypertensives with vitamin D deficiency who received an infusion of angiotensin II (AngII) before and after 1month of vitamin D3 therapy. Protocol 2 evaluated obese subjects with type 2 diabetes who also received AngII. Adipocytokines and inflammatory markers were measured before and after vitamin D3 therapy, and also before and after infusions of AngII.Vitamin D3 therapy significantly raised 25(OH)D and 1,25(OH)2D concentrations, and lowered parathyroid hormone, but had no effect on concentrations of adiponectin, resistin, leptin, IL-6, PAI-1, urinary TGF?1, or HOMA-IR. AngII infusions, despite significant elevations in blood pressure and serum aldosterone, did not influence adipocytokine concentrations in either protocol.In contrast to prior studies conducted in healthy populations, or those that could not control major regulators of the RAAS or adipocytokines, we observed that robust modulations in calcium- and sodium-regulatory hormones did not influence adipocytokines or inflammation in obesity or diabetes. Adipose-tissue physiology in these conditions may alter the hormonal regulation of inflammatory parameters.

Abstract

Diabetes is a disease of abnormal glucose homeostasis characterized by chronic hyperglycemia and a broad array of consequent organ damage. Because normal glucose homeostasis is maintained by a complex interaction between behavior (feeding and physical activity) and metabolic activity that is modulated by inter-organ signaling through secreted factors, disease modeling in vitro is necessarily limited. In contrast, in vivo studies allow complex metabolic phenotypes to be studied but present a barrier to high throughput studies. Here we present the development of a novel in vivo screening platform that addresses this primary limitation of in vivo experimentation. Our platform leverages the large secretory capacity of the liver and the hepatocyte transfection technique of hydrodynamic tail vein injection to achieve supraphysiologic blood levels of secreted proteins. To date, the utility of hydrodynamic transfection has been limited by the deleterious impact of the variable transfection efficiency inherent to this technique. We overcome this constraint by co-transfection of a secreted luciferase cDNA whose product can be easily monitored in the blood of a living animal and used as a surrogate marker for transfection efficiency and gene expression levels. To demonstrate the utility of our strategy, we screened 248 secreted proteins for the ability to enhance glucose tolerance. Surprisingly, interleukin-6 and several of its family members but not other well-recognized insulin sensitizing agents were identified as potent hypoglycemic factors. We propose this experimental system as a powerful and flexible in vivo screening platform for identifying genes that modulate complex behavioral and metabolic phenotypes.

Abstract

Disease states characterized by abnormal cellular function or proliferation frequently reflect aberrant genetic information. By revealing disease-specific DNA mutations, we gain insight into normal physiology, pathophysiology, potential therapeutic targets and are better equipped to evaluate an individual's disease risks. This review examines recent advances in our understanding of the genetic basis of adrenal cortical disease.Important advances made in the past year have included identification of KCNJ5 potassium channel mutations in the pathogenesis of both aldosterone-producing adenomas and familial hyperaldosteronism type III; characterization of phosphodiesterase 11A as a modifier of phenotype in Carney complex caused by protein kinase, cAMP-dependent, regulatory subunit, type-I mutations; the finding of 11?-hydroxysteroid dehydrogenase type I mutations as a novel mechanism for cortisone reductase deficiency; and demonstration of potential mortality benefit in pursuing comprehensive presymptomatic screening for patients with Li-Fraumeni syndrome, including possible reduction in risks associated with adrenocortical carcinoma.This research review provides a framework for the endocrinologist to maintain an up-to-date understanding of adrenal cortical disease genetics.

Abstract

Diabetes is a pathological condition characterized by relative insulin deficiency, persistent hyperglycemia, and, consequently, diffuse micro- and macrovascular disease. One therapeutic strategy is to amplify insulin-secretion capacity by increasing the number of the insulin-producing ? cells without triggering a generalized proliferative response. Here, we present the development of a small-molecule screening platform for the identification of molecules that increase ?-cell replication. Using this platform, we identify a class of compounds [adenosine kinase inhibitors (ADK-Is)] that promote replication of primary ? cells in three species (mouse, rat, and pig). Furthermore, the replication effect of ADK-Is is cell type-selective: treatment of islet cell cultures with ADK-Is increases replication of ? cells but not that of ? cells, PP cells, or fibroblasts. Short-term in vivo treatment with an ADK-I also increases ?-cell replication but not exocrine cell or hepatocyte replication. Therefore, we propose ADK inhibition as a strategy for the treatment of diabetes.

Abstract

Erdheim-Chester disease (ECD) is a rare, systemic, non-familial histiocytic disorder, first described by Jakob Erdheim and William Chester in 1930. Most patients have multiple sites of involvement at presentation. The most common site of involvement is the long bones of the axial skeleton, which is seen almost universally, followed by the nervous system, heart, lungs, orbit and retroperitoneum, which are seen in up to 50% of cases. Cutaneous involvement is rarely a presenting symptom of ECD, with two reported cases in the English literature. The diagnosis of ECD is rarely made by skin biopsy because of the relative rarity of cutaneous involvement as a presenting feature, and also perhaps because of the difficulty in distinguishing the histopathological appearance from potential mimics. The importance of distinguishing ECD from other cutaneous disorders with similar pathology lies in the implications for both treatment and prognosis. ECD is an aggressive, often fatal disorder, with death from disease occurring in greater than 50% of patients.

Abstract

Transforming growth factor-betas (TGF-beta) are secreted as inactive complexes containing the TGF-beta, the TGF-beta propeptide, also called the latency-associated protein (LAP), and the latent TGF-beta binding protein (LTBP). Extracellular activation of this complex is a critical but incompletely understood step in TGF-beta regulation. We have investigated the role of LTBP in modulating TGF-beta generation by the integrin alphaVbeta6. We show that even though alphavbeta6 recognizes an RGD on LAP, LTBP-1 is required for alphaVbeta6-mediated latent TGF-beta activation. The domains of LTBP-1 necessary for activation include the TGF-beta propeptide-binding domain and a basic amino acid sequence (hinge domain) with ECM targeting properties. Our results demonstrate an LTBP-1 isoform-specific function in alphaVbeta6-mediated latent TGF-beta activation; LTBP-3 is unable to substitute for LTBP-1 in this assay. The results reveal a functional role for LTBP-1 in latent TGF-beta activation and suggest that activation of specific latent complexes is regulated by distinct mechanisms that may be determined by the LTBP isoform and its potential interaction with the matrix.

Abstract

The mechanisms by which latent transforming growth factor beta (TGFbeta) is converted to the active cytokine are largely unknown. Here we present a genetic screen that combines retroviral mutagenesis and cDNA expression cloning to reveal proteins involved in the extracellular regulation of latent TGFbeta activation. The screen employs a cell line engineered to express green fluorescent protein (GFP) in response to TGFbeta. The cells produce their own latent TGFbeta. Therefore, after transduction with a retroviral cDNA library that contains an insert for an activator of latent TGFbeta, cells expressing the activator are GFP-bright. These cells are enriched by fluorescence-activated cell sorting and grown as individual clones. The isolated clones are cocultured with a second TGFbeta reporter cell line that produces luciferase in response to TGFbeta. Cells that have acquired the ability to activate latent TGFbeta induce luciferase expression in the absence but not in the presence of neutralizing antibodies to TGFbeta. The activator expressed by the positive clones can be identified by retrieval of the retrovirus cDNA insert.

Abstract

Epithelial-mesenchymal transformation (EMT), the process by which epithelial cells are converted into motile, invasive mesenchymal cells, is critical to valvulogenesis. Transforming growth factor-beta3 (TGF-beta3), an established mediator of avian atrioventricular (AV) canal EMT, is secreted as a latent complex. In vitro, plasmin-mediated proteolysis has been shown to release active TGF-betas from the latent complex. Annexin II, a co-receptor for tissue plasminogen activator (tPA) and plasminogen, promotes cell-surface generation of the serine protease plasmin. Here, we show that annexin II-mediated plasmin activity regulates release of active TGF-beta3 during chick AV canal EMT. Primary embryonic endocardial-derived cells express annexin II which promotes plasminogen activation in vitro. Incubation of heart explant cultures with either alpha(2)antiplasmin (alpha(2)AP), a major physiological plasmin inhibitor, or anti-annexin II IgG, blocked EMT by approximately 80%, and 50%, respectively. Anti-annexin II IgG-mediated inhibition of EMT was overcome by the addition of recombinant TGF-beta3. Upon treatment with anti-annexin II IgG or alpha(2)AP, conditioned medium from heart explant cultures showed absence of the active fragment of TGF-beta3 by Western blot analysis and a approximately 50% decrease in TGF-beta specific bioactivity. Our results suggest that annexin II-mediated plasmin activity regulates the release of active TGF-beta during cardiac valve development in the avian heart.

Abstract

TGFbeta is secreted as part of a latent complex that is targeted to the extracellular matrix. A variety of molecules, 'TGFbeta activators,' release TGFbeta from its latent state. The unusual temporal discontinuity of TGFbeta synthesis and action and the panoply of TGFbeta effects contribute to the interest in TGF-beta. However, the logical connections between TGFbeta synthesis, storage and action are obscure. We consider the latent TGFbeta complex as an extracellular sensor in which the TGFbeta propeptide functions as the detector, latent-TGFbeta-binding protein (LTBP) functions as the localizer, and TGF-beta functions as the effector. Such a view provides a logical continuity for various aspects of TGFbeta biology and allows us to appreciate TGFbeta biology from a new perspective.

Abstract

Latent transforming growth factor-beta (TGF-beta) binding protein (LTBP)-1, which is easily secreted, has been shown to enhance the secretion of TGF-beta. Here we show that another member of the LTBP family, LTBP-3, is not secreted by several cell types, but secretion occurs after coexpression with TGF-beta. The secretion of LTBP-3 requires complexing of LTBP-3 with Cys33 of the TGF-beta propeptide.

Abstract

Transforming growth factors-beta (TGFbeta1, 2 and 3) are secreted in a complex with their propeptides (latency-associated peptide 1 (LAP1), 2 and 3). TGFbeta signaling requires the dissociation of LAP and TGFbeta, a process termed latent TGFbeta activation. This process is a critical but incompletely understood step in the regulation of TGFbeta function. In particular, the extent to which activation mechanisms differ among the three TGFbeta isoforms is relatively unexplored. We show here that alphaVbeta6 binds and activates latent TGFbeta3.

Abstract

The latent transforming growth factor-beta-binding protein-1 (LTBP-1) belongs to a family of extracellular glycoproteins that includes three additional isoforms (LTBP-2, -3, and -4) and the matrix proteins fibrillin-1 and -2. Originally described as a TGF-beta-masking protein, LTBP-1 is involved both in the sequestration of latent TGF-beta in the extracellular matrix and the regulation of its activation in the extracellular environment. Whereas the expression of LTBP-1 has been analyzed in normal and malignant cells and rodent and human tissues, little is known about LTBP-1 in embryonic development. To address this question, we used murine embryonic stem (ES) cells to analyze the appearance and role of LTBP-1 during ES cell differentiation. In vitro, ES cells aggregate to form embryoid bodies (EBs), which differentiate into multiple cell lineages. We analyzed LTBP-1 gene expression and LTBP-1 fiber appearance with respect to the emergence and distribution of cell types in differentiating EBs. LTBP-1 expression increased during the first 12 d in culture, appeared to remain constant between d 12 and 24, and declined thereafter. By immunostaining, fibrillar LTBP-1 was observed in those regions of the culture containing endothelial, smooth muscle, and epithelial cells. We found that inclusion of a polyclonal antibody to LTBP-1 during EB differentiation suppressed the expression of the endothelial specific genes ICAM-2 and von Willebrand factor and delayed the organization of differentiated endothelial cells into cord-like structures within the growing EBs. The same effect was observed when cultures were treated with either antibodies to TGF-beta or the latency associated peptide, which neutralize TGF-beta. Conversely, the organization of endothelial cells was enhanced by incubation with TGF-beta 1. These results suggest that during differentiation of ES cells LTBP-1 facilitates endothelial cell organization via a TGF-beta-dependent mechanism.

Abstract

Productive interaction of a T lymphocyte with an antigen-presenting cell results in the clustering of the T-cell antigen receptor (TCR) and the recruitment of a large signalling complex to the site of cell-cell contact. Subsequent signal transduction resulting in cytokine gene expression requires the activation of one or more of the multiple isoenzymes of serine/threonine-specific protein kinase C (PKC). Among the several PKC isoenzymes expressed in T cells, PKC-theta is unique in being rapidly recruited to the site of TCR clustering. Here we show that PKC-theta is essential for TCR-mediated T-cell activation, but is dispensable during TCR-dependent thymocyte development. TCR-initiated NF-kappaB activation was absent from PKC-theta(-/-) mature T lymphocytes, but was intact in thymocytes. Activation of NF-kappaB by tumour-necrosis factor alpha and interleukin-1 was unaffected in the mutant mice. Although studies in T-cell lines had suggested that PKC-theta regulates activation of the JNK signalling pathway, induction of JNK was normal in T cells from mutant mice. These results indicate that PKC-theta functions in a unique pathway that links the TCR signalling complex to the activation of NF-kappaB in mature T lymphocytes.